Identification of neuropeptide s antagonists: Structure-Activity relationship studies, x-ray crystallography, and in vivo evaluation

Article abstract of DOI:10.1021/cn500113c

Modulation of the neuropeptide S (NPS) system has been linked to a variety of CNS disorders such as panic disorder, anxiety, sleeping disorders, asthma, obesity, PTSD, and substance abuse. In this study, a series of diphenyltetrahydro-1H-oxazolo[3,4-α]pyr

Full text of DOI:10.1021/cn500113c

Research Article
pubs.acs.org/chemneuro
Identification of Neuropeptide S Antagonists: Structure−Activity
Relationship Studies, X‑ray Crystallography, and in Vivo Evaluation
Carla Hassler,† Yanan Zhang,† Brian Gilmour,† Tyler Graf,† Timothy Fennell,† Rodney Snyder,†
Jeffrey R. Deschamps,§ Rainer K. Reinscheid,‡ Celia Garau,‡ and Scott P. Runyon*,†
†Research Triangle Institute, Post Office Box 12194, Research Triangle Park, North Carolina 27709-2194, United States
‡Department of Pharmaceutical Sciences, University of California, Irvine, 2214 Natural Sciences I, Mail Code: 3958, Irvine, California
92697-3958, United States
§Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6930, 4555 Overlook Avenue SW,
Washington, DC 20375, United States
S
* Supporting Information
ABSTRACT: Modulation of the neuropeptide S (NPS) system has been
linked to a variety of CNS disorders such as panic disorder, anxiety, sleeping
disorders, asthma, obesity, PTSD, and substance abuse. In this study, a series of
diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-ones were synthesized
and evaluated for antagonist activity at the neuropeptide S receptor. The
absolute configuration was determined by chiral resolution of the key synthetic
intermediate, followed by analysis of one of the individual enantiomers by X-ray
crystallography. The R isomer was then converted to a biologically active
compound (34) that had a Ke of 36 nM. The most potent compound displayed
enhanced aqueous solubility compared with the prototypical antagonist SHA-
68 and demonstrated favorable pharmacokinetic properties for behavioral
assessment. In vivo analysis in mice indicated a significant blockade of NPS induced locomotor activity at an ip dose of 50 mg/kg.
This suggests that analogs having improved drug-like properties will facilitate more detailed studies of the neuropeptide S
receptor system.
KEYWORDS: Neuropeptide S, structure−activity relationship, NPSR
obesity,14,15 PTSD,16,17 and importantly, substance
he discovery and evaluation of numerous neuropeptides is
Tbeginning to broaden our understanding of how abuse.18−21 NPSR activation has been shown to modulate
reward circuits and is expressed in brain areas associated with
reward processing such as the ventral tegmental area (VTA),
amygdala and substantia nigra.4 Microdialysis studies have
demonstrated that central NPS administration stimulates
dopamine release in the medial prefrontal cortex,22 and local
intra-VTA microinjections of NPS enhance dopamine release in
the nucleus accumbens.23 The latter study specifically links
NPS to the mesolimbic dopamine system, which is presumed to
be a major neuronal pathway modulating reward. NPS has also
been shown to increase cocaine self-administration in a
corticotrophin releasing factor (CRF) dependent manner.24
Blockade of the NPSR using small molecule antagonists
attenuates both drug-seeking and drug-taking behaviors.20,25
The prototypical NPS antagonist SHA-68 (1) shows selective
antagonism at the human NPSR at an in vitro potency of 13.7
nM and has been reported to prevent the arousal promoting
and anxiolytic-like effects elicited by NPS.26,27 More recently it
was found that in rats, peripheral administration of 1 reduced
endogenous peptides regulate a variety of important biological
processes. One neuropeptide that is demonstrating a number of
important modulatory roles in the central nervous system
(CNS) is neuropeptide S (NPS). NPS and its receptor (NPSR)
was originally identified in 2002 by Sato et al.,1 but Xu and
colleagues initiated the research to delineate the in vivo role(s)
of NPS in 2004.2 NPS is a 20 amino acid peptide that functions
as an agonist through activation of Gq or Gs protein coupled
pathways.2 Defining both receptor and peptide localization in
the CNS provided the first indications of NPS function. In situ
hybridization showed that NPSR mRNA was expressed widely
throughout the CNS.3 However, peptide precursor mRNA was
limited to the locus ceruleus (LC) area and the parabrachial
nucleus of the brainstem.4 Based on these observations, NPS
was hypothesized to be involved in a number of CNS functions.
The human NPSR has two splice variants with a number of
polymorphisms. One that has been well characterized occurs at
position 107 and generates two variants of the receptor that
differ significantly in their pharmacology5 and have been linked
to panic disorder6,7 and fear processing.8 Since the discovery of
NPS in 2002, modulation of the NPS system has also been
linked to anxiety,9,2,10,11 sleeping disorders,2,12 asthma,13
Received: May 21, 2014
Revised: June 24, 2014
Published: June 25, 2014
© 2014 American Chemical Society
731
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
Figure 1. Representative classes of NPS antagonists.
a
Scheme 1. Synthesis of Key Intermediate 37
a
Reagents and conditions: (a) PhLi, THF, −78 °C; (b) H2 (46 psi), 10% Pd/C, 5% Pd/BaSO4, EtOH; (c) Boc2O, DMAP, NEt3, THF; (d) TFA,
DCM.
conditioned reinstatement of cocaine seeking in two stud-
ies,25,20 yet it was found in one study to reduce cocaine self-
administration but only with a concomitant reduction in food
self-administration at doses up to 30 mg/kg, ip.20
self-administration at lower doses (10−20 mg/kg, ip) without
affecting food self-administration. These results indicate that 2
has better in vivo efficacy for reducing cocaine self-
administration compared with 1. Interestingly, 2 (Ke = 109
nM) has roughly 8-fold reduction in antagonist potency versus
1 (13.9 nM) at the NPSR 107I variant when tested in the
calcium mobilization assay.27 One distinct structural difference
between these compounds is the ability of 2 to be partially
ionized at physiological pH and thus more water-soluble.
Therefore, the observed difference in efficacy between the two
antagonists could be due to differences in drug-like properties.
We herein report the further evaluation of RTI-118,
including the in vitro and in vivo pharmacokinetic properties,
as well as additional SAR studies in an effort to shed light on
the possible mechanisms for the behavioral differences between
the two series. Although five structural classes of small molecule
NPS antagonists are currently known, a comprehensive
knowledge of the structure−activity relationships is still
somewhat limited and a more detailed understanding of the
molecular requirements for antagonist activity at the NPSR is
required. The continued development of small molecules
having improved potency and better drug-like properties than 1
may enable a better understanding of the NPS receptor system.
In this study, we describe steps toward the development of a
comprehensive pharmacophore with a bias toward increasing
aqueous solubility and drug-like properties. Continued develop-
ment of molecules that have improved drug-like properties will
enable more detailed studies of the NPSR as an emerging target
for a variety of CNS related disorders.
Several other small molecule NPSR antagonists have recently
been reported, primarily by several large pharmaceutical
companies, most of which are yet to be evaluated in vivo.
For instance, Merck Research Laboratories identified two
scaffolds through high-throughput screening (HTS) that have
high potency at the NPSR using a calcium mobilization assay.
Quinolinone scaffold 3 had a potency of 18 nM and was found
to have acceptable drug-like properties,28 and the second series
contained a tricyclic imidazole that displayed potencies in the
40−80 nM range.29 In addition, GlaxoSmithKline identified a
highly potent naphthopyranopyrimidine scaffold 4.30 Finally,
another small molecule scaffold identified to date is
NCGC00185684 (5), which resulted from an NIH Molecular
Libraries HT screen.31 Compound 5 has good potency in both
calcium mobilization and cAMP assays at 1 and 45 nM,
respectively, and has recently been shown to reduce operant
self-administration of alcohol at 1 mg/kg ip.
Our laboratory has developed a series of NPS antagonists
based on the structure of SHA-68 and has recently
demonstrated that one of these compounds, RTI-118 (2,
Figure 1) blocks cocaine self-administration, cue, stress, and
drug induced reinstatement.20 Interestingly, compounds 1 and
2 both reduced cue-induced reinstatement of extinguished
cocaine self-administration in rats at similar doses (30 mg/kg),
yet only 2 selectively blocked cocaine self-administration.
Although at a higher dose of 50 mg/kg 1 was able to reduce
cocaine self-administration, it also decreased food-maintained
responding, suggesting a nonselective impairment of operant
behavior. However, 2 was able to selectively reduce cocaine
CHEMISTRY
■
The synthesis of all target compounds in this study was based
on modifications to a central core scaffold. The synthesis of
732
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
a
Scheme 2. Synthesis of Target Molecules Derived from Isocyanate or Acid Chloride Addition
a
Reagents and Conditions: (a) R-isocyanate, CH2Cl2, rt; (b) acid chloride, TEA, 0 °C; (c) CH3NH2, DMF, KHCO3, rt; (d) (CH3)2NH, DMF,
KHCO3, rt.
a
Scheme 3. Preparation of Target Molecules Using Triphosgene Conditions
a
Reagents and conditions: (a) R-amine, (CCl3O)2CO, TEA, THF, 0 °C to rt; (b) TFA, DCM; (c) HCOH, NaH(OAc)3, 1,2-DCE.
a
Scheme 4. Identification of the Active Enantiomer of Key Intermediate 37
a
Reagents and Conditions: (a) chiral HPLC; b) R-camphorsulfonic acid, MeOH; (c) 4-fluorobenzyl isocyanate.
core scaffold 37 was initiated through the condensation of 2
equiv of phenyl lithium with commercially available ethyl 1,4-
dibenzylpiperazine-2-carboxylate (Scheme 1). The resulting
alcohol 35 was debenzylated using 5% palladium on barium
733
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
sulfate in ethanol to afford piperazine 36. Cyclization of 36 was
accomplished using tert-butyl dicarbonate, triethylamine, and
dimethylaminopyridine in tetrahydrofuran.32 This reaction
required at least 1 equiv of dimethylaminopyridine to provide
clean product. The resulting cyclic tert-butyl carbamate
intermediate was deprotected with trifluoroacetic acid in
dichloromethane to provide core scaffold 37 in good overall
yield.
One of the primary goals of this research was to identify
substituents that would confer improved aqueous solubility.
Rapid condensation of a number of commercially available
isocyanates with core scaffold 37 in dichloromethane provided
compounds 1, 6, 16−21, 40, and 41 in good yield (Scheme 2).
Displacement of the alkyl chlorides 40 and 41 with potassium
carbonate, methyl amine, or dimethyl amine in dimethylforma-
mide provided target molecules 22−24, respectively, in
moderate yield. Condensation of 3-(piperidin-1-yl)propanoic
acid chloride with core scaffold 37 provided amide analog 25 in
excellent yield.
Additional analogs containing basic amines were synthesized
using a general method that incorporated the appropriate
amine, triphosgene, and triethylamine in tetrahydrofuran.
Compounds 2, 7−13, and 26 were prepared from 37 using
triphosgene, triethylamine, and piperidine ethyl amine,
alternately substituted pyridine methyl amines, alternately
substituted pyridine ethyl amines, tert-butyl 2-(aminomethyl)-
phenylcarbamate, and 1-methylpiperidin-4-amine, respectively
(Scheme 3). Deprotection of 13 using trifluoroacetic acid in
dichloromethane provided 14. Reductive amination using
sodium triacetoxyborohydride in 1,2-dichloroethane with 1
equiv of formaldehyde afforded a mixture of mono and
dialkylated products from which 15 was isolated. Target
compounds 27−30 were obtained using analogous methods to
that described for compound 14.
The identification of the active enantiomer of core scaffold
37 was performed using a combination of chiral HPLC and X-
ray crystallography. Racemic 37 was subjected to chiral HPLC
to afford enantiomers 38 and 39 (Scheme 4). The R-
camphorsulfonic acid salt of 38 was prepared from R-
camphorsulfonic acid in methanol. The resulting salt 38a was
subjected to X-ray analysis, and the chirality was determined to
be S (Figure 2). Compounds 31 and 32 were then prepared
from 38 and 39, respectively, using 4-fluorobenzyl isocyanate in
tetrahydrofuran. Compounds 33 and 34 were isolated via
separation of 2 by chiral HPLC.
Figure 2. X-ray structure of the inactive enantiomer 38a.
improved potency will provide additional tools to study the
NPSR.
Considering the challenges of formulation and drug delivery
for analogs like 1, our laboratory set out to interrogate the
tolerances to scaffold modification with the goal of improving
aqueous solubility and receptor potency. Compounds were
analyzed for agonist activity, and when they displayed no
intrinsic activity, antagonist activity at the NPSR was measured
using a functional calcium mobilization assay in stably
transfected CHO-K1 cells. Each clone individually expressed
one of the two predominant polymorphisms, 107I or 107N.
The apparent constant Ke was determined by running EC50
curves of the agonist, NPS, with and without the test
compounds to measure the rightward shift of the agonist
curve. In keeping with literature reports, most compounds were
more potent at the 107I variant of the NPSR ranging from 5.5-
fold more potent to 0.59-fold. On average compounds were 1−
2-fold more potent at the 107I variant. Ke values throughout
the results section refer to results obtained using the 107I
construct unless specifically noted.
The benzyl urea functions of 1 and 6 have previously been
shown as preferred substituents for antagonist potency.27 Based
on this information, we introduced a series of benzylpyridines
and pyridinylethyl ureas to enhance solubility. Substitution of 1
with a 2-pyridyl (7) reduced potency by 19-fold, while both the
3-pyridyl (8) and 4-pyridyl (9) also displayed a 19- and 30-fold
reduction in potency, respectively, versus 1 (Table 1). This
suggests that either increased polarity or electron density is not
tolerated at this position. Elongation of the linker to ethyl
further reduced antagonist potency. Compounds 10−12 had
respective potencies of 945, 579, and 1157 nM as antagonists.
In another attempt to provide compounds that could remain
protonated in a suitable in vivo formulation, 2-aminobenzyl
analogs were evaluated. N-Boc protected 13 had a limited effect
on potency suggesting that this position could be modified with
a variety of substituents and retain activity. Deprotection of 13
afforded free amine 14 (32 nM) that was only 2-fold reduced in
activity from 1. Monomethylation provided 15 having a Ke of
90 nM. Based on the data from Table 1, it appears that the
RESULTS AND DISCUSSION
■
One of the prototypical NPSR antagonists, SHA-68 (1), has
been studied in a number of in vivo behavioral and in vitro
biological assay systems. Pharmacokinetic studies have been
performed on 1 and indicate that a significant component of
the ip dose enters the CNS,26 yet it behaves differently from 2
in vivo.20 In order to provide a potential basis for this
discrepancy, the aqueous solubility of 1 and 2 was evaluated.
Aqueous solubility for 2 improved by a factor of 4200 at pH 3
and 3600 at pH 7 compared with 1 as determined by filtration
through a 0.4 μm polycarbonate membrane in aqueous buffer
(Millipore MultiScreen PCF 96 well filter plate).33 Solubility at
pH 3 and 7 for compound 1 is 0.01 and 0.01 μM and that for
compound 2 is 42 and 36 μM, respectively. The enhanced
solubility of 2 compared with 1 is quite striking and suggests
the in vivo differences between 1 and 2 may be related to drug-
like properties. Analogs that retain this property and have
734
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
Table 1. Potency of Benzyl Urea Analogs of SHA-68 (1) in
CHOK1 Cells Expressing Human NPSR Isoforms 107I and
107N
Table 2. Potency of Alkyl Substituted Urea Analogs of SHA-
68 in CHOK1 Cells Expressing Human NPSR Isoforms 107I
and 107N: Alkyl Substitution of the Urea to Determine
Optimal Substituent Size
to isopropyl (17, 953 nM), to propyl (18, 201 nM), and then
butyl (19, 33 nM) indicated that a benzylic moiety was not a
requirement for optimal potency and alkyl substituted analogs
could be prepared with comparable potency to 1. Further
elongation of the alkyl chain to pentyl (20, 61 nM) and hexyl
(21, 184 nM) started to reduce potency from the optimal
activity of butyl (19). This suggested that a lipophilic pocket of
limited size accommodated aliphatic and benzylic groups. Once
compound 19 was identified as the most potent alkyl analog,
we set out to evaluate how incorporating amine groups would
be tolerated. Compound 22 is an amino substituted analog of
19 but displays a 21-fold reduction in activity. Increasing the
distance of amine from the urea by one carbon (23) slightly
improved potency compared with 22, but 23 remained 14-fold
less potent than the n-butyl analog 22. Methylation of 23
reduced activity to a Ke of 782 nM for dimethylated analog
(24). This data indicates that substituted amino groups can be
tolerated as in the case of 2 (Ke = 109 nM) but the disposition
of the polar group is critical to activity.
Considering that compound 2 displayed moderate potency
in earlier studies, we set out to define the receptor tolerances
through altering the placement of ionizable amino groups
within cyclic substituents (Table 3). Converting the urea
functionality to an amide was performed to determine whether
a series of commercial amides could be evaluated more rapidly.
Replacement of the urea led to reduced potency for 25 having a
Ke of 394 nM. This data supports earlier findings where
benzylic aryl ring is sensitive to alteration and that compounds
with pyridyl substitution are not well tolerated.
Previous studies modifying the SHA-68 scaffold (1) indicated
that benzylic substituents are preferred but that aliphatic
substituents can also lead to analogs with moderate potency.27
We thus set out to evaluate the size requirements for aliphatic
substituents in place of the benzyl group on 1 (Table 2). Once
the size limitations of the hypothetical binding site were
determined, we could then evaluate tolerances to incorporation
of solubilizing aliphatic amine groups. Progressive substitution
with larger aliphatic moieties starting from ethyl (16, 2270 nM)
735
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
appended with a large variety of substituents. The ability to
further explore SAR using the known biologically active isomer
of a synthetic intermediate is of significance. Thus, the
intermediate amine 37 was separated into two isomers 38
and 39 using chiral HPLC. The chirality of these compounds
was elucidated by preparing the R-camphorsulfonic acid salt of
one of the isomers, 38, and performing an X-ray study on the
resulting salt 38a. Based on this study 38a had the S
configuration. The corresponding 4-fluorobenzyl ureas were
then prepared from 38 (S) and 39 (R), respectively, and tested
in the calcium mobilization assay. Compound 32 showed a Ke
of 5.1 nM, whereas 31 was inactive up to 5 μM, confirming that
the R configuration is required for activity at the NPS receptor
(Table 4). Similarly, the individual isomers of the piperidylethyl
analog 33 and 34 were isolated, and only one isomer, 34,
displayed activity (36 nM) at the NPS receptor.
Table 3. Potency of Urea Analogs of SHA-68 in CHOK1
Cells Expressing Human NPSR Isoform 107I: Urea
Substituents Designed to Enhance Aqueous Solubility
Table 4. Potency of Individual Enantiomers of Lead
Compounds 1 and 2 in CHOK1 Cells Expressing Human
NPSR Isoform 107I
methylation of the urea nitrogen on 1 significantly reduced
potency.27 Compound 26 replaced the piperidinylethyl in 2
with an N-methylpiperidine that displayed significantly reduced
activity (2091 nM) compared with 2. Both the N-Boc protected
and free amino pyrrolidines 27 and 28 also displayed reduced
activity whereas pyrrolidine analogs 29 and 30 displayed
potencies of 3384 nM and 510 nM, respectively. These data
combined with results for the acyclic analogs 16−21 suggest
that the receptor site prefers lipophilic substituents of specific
size and that amides display reduced activity compared with
ureas.
The importance of the chirality in 1 has been demonstrated
where only the R isomer was active.34 Similarly, only one
bioactive enantiomer was isolated from the racemic mixture of
NPS antagonist 3.28 The original chiral study of 1 utilized a
multistep synthesis that relied on diastereomeric separation.
Considering that one enantiomer is known to retain activity we
set out to identify the active enantiomer of the synthetically
common intermediate 37 that could be used to rapidly evaluate
a number of chiral analogs with this class of compounds. The
chiral intermediate described in this study (37) can be readily
In order to get a more detailed understanding of on-target
effects in vivo, compound 34 was further evaluated for its
pharmacokinetic properties (Figure 3). Following an ip dose,
34 was rapidly absorbed into systemic circulation, with peak
plasma concentration observed at 15 min postdose. After a
distribution phase, plasma concentrations declined with an
apparent half-life of 33.9 min. Brain concentrations of 34 were
measurable as early as 15 min postdose (the first sampling time
point), with peak brain concentration observed at 15 min
postdose. Compound 34 was eliminated from the brain with an
apparent half-life of 143 min. The overall brain to plasma AUC
ratio, as determined by AUClast or AUC0‑inf ratio, was 0.14 or
0.29, respectively. This study provides a preliminary assessment
of the brain penetration of 34 in mice after an ip dose of 5 mg/
kg. At 90 min postdose, brain concentrations were approx-
736
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
Figure 3. Pharmacokinetic analysis of compound 34 in brain and
plasma; 5 mg/kg dose. Sample size, five animals per time point. Data
points represent the mean, and error bars represent standard deviation.
imately 60 nM indicating sufficient brain penetration for NPS
receptor modulation considering 34 has a Ke of 36 nM.
In Vivo Activity of 34. NPS is known to induce
hyperlocomotion in mice after central administration.2 To
test whether this central effect of NPS can be antagonized by
34, we recorded distance traveled at 5 min intervals over 90
min in mice that had received injections of vehicle or 34 (5 and
50 mg/kg ip) and subsequently received central administrations
of either vehicle or 1 nmol of NPS icv. As shown in Figure 4,
analysis of cumulative activity data revealed that injections of
NPS + vehicle increased the cumulative distance traveled by the
animals in a statistically significant manner. Compound 34
alone at 50 mg/kg did not modify per se the animal behavior
compared with the control group (p = 0.8738, unpaired
Student’s t test). When challenged with NPS, 34 at a dose of 5
mg/kg resulted in a nonsignificant reduction (approximately
20%) of the distance traveled as compared with mice receiving
NPS + vehicle injections (p = 0.3515, unpaired Student’s t
test). At the highest dose (50 mg/kg), 34 significantly
attenuated (approximately 60%) the effects of 1 nmol of
NPS, demonstrating that 34 (50 mg/kg) was effective at least
partially antagonizing NPS-induced activity. Statistically sig-
nificant effects were obtained with 50 mg/kg, although the
antagonist did not fully prevent the effects elicited by the
peptide. Together, these data demonstrate that 34 behaves as
an NPSR antagonist in vivo and is centrally acting after
peripheral administration.
Figure 4. Effects of 34 on motor activity in mice. Mice had been
habituated for 60 min before injection. Compound 34 was dissolved in
10% Cremophor EL (vehicle) and injected (ip) 10 min before NPS or
vehicle (PBS, 0.1% BSA) were injected centrally (icv). Group sizes:
Veh + Veh, n = 8; Veh + NPS, n = 7; 34 + PBS, n = 7; 34 (50 mg/kg)
+ NPS (1 nmole), n = 8; 34 (5 mg/kg) + NPS (1 nmole), n = 9. (A)
Time course of the distance traveled over 90 min. (B) Total distance
traveled during the 90 min observation period. *, p < 0.001, Veh + Veh
versus Veh + NPS; #, p < 0.001 Veh + NPS versus NPS + 34 (50 mg/
kg) (Students t test).
CONCLUSIONS
■
A series of N-substituted diphenyltetrahydro-1H-oxazolo[3,4-
α]pyrazin-3(5H)-ones have been synthesized and evaluated for
antagonism of the NPS receptor system. The receptor tolerance
to both size and polarity of these analogs at several locations
were evaluated. The most potent antagonists possessed simple
aliphatic ureas such as N-butyl (19) or ureas having basic amine
containing substituents that were substituted with hydrophobic
groups such as piperidinoethyl (34). Incorporation of
protonatable nitrogen improved the aqueous solubility of this
series while maintaining NPS receptor potency. Resolution of
the key intermediate for the synthesis of these analogs and
identification of the absolute configuration of the bioactive
isomer now affords the opportunity to rapidly obtain a number
of chiral analogs for future study. In vivo behavioral analysis of
the most potent compound demonstrated the ability of one
such compound (34) to significantly reduce NPS stimulated
locomotor activity at a dose of 50 mg/kg. The analogs
described in this study provide additional detail as to the
requirements for biological activity at the NPSR and represent
improved tools to study the NPSR in vivo. The improvement of
aqueous solubility opens up possibilities not permitted with less
soluble analogs such as iv administration and microinjections
into discrete brain regions using an unmodified aqueous buffer
system. Identification of additional analogs having improved
properties could ultimately lead to pharmacotherapies to treat a
variety of CNS disorders.
737
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
= 4.52 Hz, 2 H), 1.58 (m, J = 4.90 Hz, 4 H), 2.18 (dd, J = 13.19, 11.30
Hz, 1 H), 2.37−2.55 (m, 6 H), 2.84 (td, J = 12.72, 3.58 Hz, 1 H), 3.08
(td, J = 12.62, 3.77 Hz, 1 H), 3.31 (quin, J = 5.18 Hz, 2 H), 3.73−3.97
(m, 3 H), 4.43 (dd, J = 11.11, 3.58 Hz, 1 H), 5.47−5.74 (m, 1 H),
7.28−7.45 (m, 8 H), 7.49−7.58 (m, 2 H). ESI HRMS m/z: calcd for
C26H32N4O3 448.57, found 449.25 (M + H)+. HPLC (Synergy Hydro,
20 min) tR = 15.23 min (>99%).
METHODS
■
General. All standard reagents were commercially available.
Compounds were purified by column chromatography on a Teledyne
Isco Rf chromoatography unit and by HPLC on an Agilent-Varian
HPLC system equipped with Prostar 210 dual pumps, a Prostar 335
diode UV detector and a SEDEX75 (SEDERE, Olivet, France) ELSD
detector. The HPLC solvent system was binary, water containing 0.1%
trifluoroacetic acid (TFA) and solvent B (acetonitrile containing 0−
5% water and 0.1% TFA). A semipreparative Synergy Hydro RP 80A
C18 column (4 μm, 250 mm × 21.2 mm column; Phenomenex) or a
Gemini-NX RP C18 column (5 μm, 250 mm × 21.2 mm;
Phenomenex) was used to purify final compounds at 15 mL/min
using a linear gradient from 5% to 50% or 60% B over 30 min. The
purity of final compounds was determined using an analytical Synergy
Hydro RP80A C18 (4 μm, 250 mm × 4.60 mm column;
Phenomenex) or a Gemini-NX C18 (5 μm, 250 mm × 4.6 mm;
Phenomenex) with a linear gradient of 5%−95% solvent B over 20 or
30 min at a flow rate of 1 mL/min. Absorbance was monitored at 220
nm. Individual enantiomers were separated, and enantiomeric purity
was determined using an analytical Chiralpak-IA column (4.6 mm ×
250 mm) with isocratic conditions (hexanes/isopropanol 9/1 or
methyl-tert-butylether/ethanol 8/2 or 95/5 containing 0.1% DEA; 1
mL/min). The molecular ion of final compounds was determined
using a PE Sciex API 150 EX LC/MS system from PerkinElmer (San
Jose, California). Reactions were monitored by thin-layer chromatog-
raphy (TLC) carried out on precoated 60 Å 250 mm silica gel TLC
plates with F-254 indicator visualized under UV light and developed
N-Benzyl-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (6). An analogous procedure to that
described for 1 using benzyl isocyanate provided 6 as a white solid (31
1
mg, 86%). H NMR (300 MHz, chloroform-d) δ ppm 2.10−2.22 (m,
1 H), 2.81−2.95 (m, 1 H), 3.00−3.12 (m, 1 H), 3.59−3.70 (m, 1 H),
3.82 (dd, J = 13.00, 3.20 Hz, 1 H), 4.03 (dt, J = 13.28, 1.84 Hz, 1 H),
4.31−4.49 (m, 3 H), 4.89 (br s, 1 H), 7.24−7.42 (m, 13 H), 7.47−7.54
(m, 2 H). ESI HRMS m/z: calcd for C26H25N3O3 427.19, found
450.18 (C26H25N3O3 +Na)+.
3-Oxo-1,1-diphenyl-N-(pyridin-2-ylmethyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide (7). An analogous pro-
cedure to that described for 2 using pyridin-2-ylmethanamine provided
7 as a white powder (42 mg, 58%). 1H NMR (300 MHz, chloroform-
d) δ ppm 2.21 (dd, J = 13.19, 11.30 Hz, 1 H), 2.96 (dd, J = 13.56, 3.77
Hz, 1 H), 3.10 (dd, J = 13.19, 3.77 Hz, 1 H), 3.78−3.93 (m, 2 H), 4.04
(dd, J = 13.37, 2.45 Hz, 1 H), 4.44 (dd, J = 11.30, 3.77 Hz, 1 H),
4.48−4.68 (m, 2 H), 5.95−6.09 (m, 1 H), 7.21 (dd, J = 7.16, 5.27 Hz,
1 H), 7.27−7.44 (m, 9 H), 7.49−7.60 (m, 2 H), 7.67 (td, J = 7.63, 1.70
Hz, 1 H), 8.52 (d, J = 4.90 Hz, 1 H). ESI MS m/z: calcd for
C25H24N4O3 428.49, found 427.6 (M − H)−, 429.2 (M + H)+, 451.5
(M + Na)+. HPLC (Synergy Hydro, 20 min) tR = 14.61 min (95.0%).
3-Oxo-1,1-diphenyl-N-(pyridin-3-ylmethyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide (8). An analogous pro-
cedure to that described for 2 using pyridin-3-ylmethanamine provided
8 as white powder (52 mg, 71%). 1H NMR (300 MHz, chloroform-d)
δ ppm 2.19 (dd, J = 13.56, 11.30 Hz, 1 H), 2.95 (dd, J = 13.00, 3.20
Hz, 1 H), 3.03−3.15 (m, 1 H), 3.61−3.70 (m, 1 H), 3.82−3.93 (m, 1
H), 3.95−4.05 (m, 1 H), 4.34−4.51 (m, 2 H), 4.83 (br s, 1 H), 7.22−
7.43 (m, 10 H), 7.48−7.55 (m, 2 H), 7.64 (d, J = 7.16 Hz, 1 H), 8.51−
8.57 (m, 1 H). ESI MS m/z: calcd for C25H24N4O3 428.49, found
427.4 (M − H)−, 429.0 (M + H)+, 451.3 (M + Na)+. HPLC (Synergy
Hydro, 20 min) tR = 14.20 min (93.4%).
3-Oxo-1,1-diphenyl-N-(pyridin-4-ylmethyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide (9). An analogous pro-
cedure to that described for 2 using pyridin-4-ylmethanamine provided
9 as a white powder (47 mg, 65%). 1H NMR (300 MHz, chloroform-
d) δ ppm 2.20 (dd, J = 13.56, 11.30 Hz, 1 H), 2.89−3.02 (m, 1 H),
3.03−3.18 (m, 1 H), 3.62−3.75 (m, 1 H), 3.82−3.94 (m, 1 H), 4.02
(ddd, J = 13.38, 3.58, 1.51 Hz, 1 H), 4.31−4.51 (m, 3 H), 5.01 (br s, 1
H), 7.13−7.23 (m, 2 H), 7.24−7.44 (m, 8 H), 7.47−7.58 (m, 2 H),
8.49−8.64 (m, 2 H). ESI MS m/z: calcd for C25H24N4O3 428.49,
found 429.3 (M + H)+; 451.4 (M + Na)+, 427.5 (M − H)−, HPLC
(Synergy Hydro, 20 min) tR = 14.04 min (94.1%).
1
using ceric ammonium molybdate. H NMR spectra were recorded at
300 MHz on a Bruker Avance 300 Spectrospin instrument and are
reported as follows: chemical shift δ in ppm (multiplicity, coupling
constant (Hz), and integration). The following abbreviations were
used to explain multiplicities: s = singlet, d = doublet, t = triplet, q =
quartet, quin = quintet, m = multiplet, br = broad, dd = doublet of
doublets, dt = doublet of triplets, ddd = doublet of doublets of
doublets, ddt = doublet of doublet of triplets. Optical rotations were
determined using a Rudolph Research Autopol III automatic
polarimeter.
N-(4-Fluorobenzyl)-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo-
[3,4-a]pyrazine-7(3H)-carboxamide (1). To a solution of 37 (50 mg,
0.17 mmol) in CH2Cl2 (5 mL) at 0 °C under N2 was added 1-fluoro-4-
(isocyanatomethyl)benzene (143 μL, 0.170 mmol). The reaction
mixture was stirred at room temperature for 2 h and concentrated, and
the residue was purified by medium pressure column chromatography
(12 g f SiO2, 100% methylene chloride to 50% chloroform/methanol/
ammonium hydroxide 80/20/1) followed by semipreparative HPLC
1
to provide 1 as a white powder (29 mg, 30%). H NMR (300 MHz,
chloroform-d) δ ppm 2.17 (dd, J = 13.19, 11.30 Hz, 1 H), 2.83−3.00
(m, 1 H), 3.01−3.16 (m, 1 H), 3.63 (dd, J = 13.00, 1.70 Hz, 1 H), 3.86
(dd, J = 13.00, 2.45 Hz, 1 H), 4.03 (dd, J = 13.19, 2.26 Hz, 1 H),
4.26−4.49 (m, 3 H), 4.69−4.81 (m, 1 H), 6.95−7.09 (m, 1 H), 7.21−
7.45 (m, 11 H), 7.47−7.56 (m, 2 H). ESI MS m/z: calcd for
C26H24FN3O3 445.49, found 446.7 (M + H)+. HPLC (Synergy Hydro,
20 min) tR = 19.47 min (>99%).
3-Oxo-1,1-diphenyl-N-(2-(pyridin-2-yl)ethyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide (10). An analogous
procedure to that described for 2 using 2-(pyridin-2-yl)ethanamine
1
provided 10 as white powder (50 mg, 66%). H NMR (300 MHz,
3-Oxo-1,1-diphenyl-N-(2-(piperidin-1-yl)ethyl)tetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (2). To a solution of 37
(0.5 g, 1.70 mmol) in THF (anhydrous, 70 mL) at 0 °C under N2 was
added triphosgene (0.17 g, 0.57 mmol), and the reaction mixture was
allowed to stir at 0 °C for 5 min. To this mixture was added
triethylamine (710 μL, 5.1 mmol), and the reaction was allowed to stir
at 0 °C for 5 min. To this mixture was then added piperidine
ethylamine (240 μL, 1.67 mmol) at 0 °C, and the reaction mixture was
allowed to stir at 0 °C for 2 h and then warmed to room temperature.
The resulting suspension was concentrated to dryness and taken up in
CH2Cl2 (100 mL). The suspension was made basic with sat. aq.
NaHCO3 and extracted with CH2Cl2 (3 × 100 mL). The organic
layers were washed with brine (50 mL), dried (MgSO4), and
concentrated. The residue was purified by column chromatography
(12 g of SiO2, 100% methylene chloride to 40% chloroform/
methanol/ammonium hydroxide 80/20/1) provided 2 (470 mg, 62%)
as a white solid. 1H NMR (300 MHz, chloroform-d) δ ppm 1.48 (d, J
chloroform-d) δ ppm 2.17 (dd, J = 13.19, 11.30 Hz, 1 H), 2.84 (td, J =
12.81, 3.39 Hz, 1 H), 2.93−3.11 (m, 3 H), 3.55−3.66 (m, 2 H), 3.72−
3.87 (m, 2 H), 3.89−4.00 (m, 1 H), 4.39 (dd, J = 11.11, 3.58 Hz, 1 H),
6.30 (t, J = 4.90 Hz, 1 H), 7.12−7.21 (m, 2 H), 7.23−7.42 (m, 8 H),
7.46−7.55 (m, 2 H), 7.63 (td, J = 7.72, 1.88 Hz, 1 H), 8.43 (dt, J =
4.62, 1.46 Hz, 1 H). ESI MS m/z: calcd for C26H26N4O3 442.52, found
443.4 (M + H)+, 441.4 (M − H)−. HPLC (Synergy Hydro, 20 min) tR
= 14.76 min (97.8%).
3-Oxo-1,1-diphenyl-N-(2-(pyridin-3-yl)ethyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide (11). An analogous
procedure to that described for 2 using 2-(pyridin-3-yl)ethanamine
1
provided 11 as a white powder (28 mg, 37%). H NMR (300 MHz,
chloroform-d) δ ppm 2.14 (dd, J = 13.19, 11.30 Hz, 1 H), 2.78−2.95
(m, 3 H), 3.02 (td, J = 12.72, 3.58 Hz, 1 H), 3.34−3.53 (m, 2 H), 3.60
(d, J = 12.81 Hz, 1 H), 3.81 (dd, J = 12.81, 2.64 Hz, 1 H), 3.94 (dd, J =
13.19, 3.39 Hz, 1 H), 4.39 (dd, J = 11.30, 3.77 Hz, 1 H), 7.17−7.44
738
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
(m, 9 H), 7.46−7.56 (m, 3 H), 8.35−8.50 (m, 2 H). ESI MS m/z:
calcd for C26H26N4O3 442.52, found 441.3 (M − H)−, 443.5 (M +
H)+, 465.4 (M + Na)+. HPLC (Synergy Hydro, 20 min) tR = 14.59
min (97.9%).
mg, 93%). 1H NMR (300 MHz, chloroform-d) δ ppm 1.13 (t, J = 7.16
Hz, 3 H), 2.07−2.20 (m, 1 H), 2.81−2.96 (m, 1 H), 3.07 (td, J =
12.72, 3.58 Hz, 1 H), 3.19−3.35 (m, 2 H), 3.58−3.69 (m, 1 H), 3.84
(dd, J = 13.00, 2.45 Hz, 1 H), 4.00 (ddd, J = 13.28, 3.49, 1.32 Hz, 1
H), 4.42 (dd, J = 11.30, 3.39 Hz, 1 H), 4.50−4.58 (m, 1 H), 7.25−7.42
(m, 8 H), 7.47−7.54 (m, 2 H). ESI HRMS m/z: calcd for C21H23N3O3
365.17, found 388.16 (M + Na)+.
N-Isopropyl-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (17). An analogous procedure to that
described for 1 using i-propylisocyanate provided 17 as a viscous oil
(166 mg, 93%). 1H NMR (300 MHz, chloroform-d) δ ppm 1.10−1.21
(m, 6 H), 2.02−2.18 (m, 1 H), 2.79−2.95 (m, 1 H), 3.07 (td, J =
12.62, 3.39 Hz, 1 H), 3.59 (dt, J = 13.19, 1.70 Hz, 1 H), 3.78−4.11 (m,
3 H), 4.33 (d, J = 7.16 Hz, 1 H), 4.42 (dd, J = 11.30, 3.77 Hz, 1 H),
7.25−7.44 (m, 8 H), 7.46−7.56 (m, 2 H). ESI HRMS m/z: calcd for
C22H25N3O3 379.19, found 402.18 (M + Na)+.
3-Oxo-1,1-diphenyl-N-(2-(pyridin-4-yl)ethyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide (12). An analogous
procedure to that described for 2 using 2-(pyridin-4-yl)ethanamine
1
provided 12 as a white powder (55 mg, 72%). H NMR (300 MHz,
chloroform-d) δ ppm 2.09−2.20 (m, 1 H), 2.78−2.91 (m, 3 H), 3.01
(td, J = 12.72, 3.20 Hz, 1 H), 3.40−3.57 (m, 2 H), 3.59−3.71 (m, 1
H), 3.78 (d, J = 13.19 Hz, 1 H), 3.96 (dd, J = 13.37, 2.83 Hz, 1 H),
4.39 (dd, J = 11.11, 3.58 Hz, 1 H), 7.04−7.11 (m, 2 H), 7.19−7.44 (m,
8 H), 7.44−7.53 (m, 2 H), 8.40 (dd, J = 4.33, 1.70 Hz, 2 H). ESI MS
m/z: calcd for C26H26N4O3 442.52, found 441.5 (M − H)−, 443.5 (M
+ H)+, 465.3 (M + Na)+. HPLC (Synergy Hydro, 20 min) tR = 14.55
min (90.6%).
tert-Butyl-2-((3-oxo-1,1-diphenylhexahydro-1H-oxazolo[3,4-a]-
pyrazine-7-carboxamido)methyl)-phenylcarbamate (13). An analo-
gous procedure to that described for 2 using tert-butyl-2-
(aminomethyl)phenylcarbamate provided 13 as a viscous oil (75 mg,
3-Oxo-1,1-diphenyl-N-propyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (18). An analogous procedure to that
described for 1 using n-propylisocyanate provided 18 as a viscous oil
(166 mg, 93%). 1H NMR (300 MHz, chloroform-d) δ ppm 0.91 (t, J =
7.54 Hz, 3 H), 1.46−1.59 (m, 2 H), 2.07−2.20 (m, 1 H), 2.83−2.95
(m, 1 H), 3.07 (td, J = 12.62, 3.39 Hz, 1 H), 3.18 (tdd, J = 7.25, 7.25,
5.46, 1.88 Hz, 2 H), 3.63 (dt, J = 13.19, 1.88 Hz, 1 H), 3.84 (dd, J =
12.81, 2.64 Hz, 1 H), 4.01 (ddd, J = 13.28, 3.67, 1.51 Hz, 1 H), 4.42
(dd, J = 11.30, 3.39 Hz, 1 H), 4.55 (t, J = 5.46 Hz, 1 H), 7.23−7.44
(m, 8 H), 7.47−7.56 (m, 2 H). ESI HRMS m/z: calcd for C22H25N3O3
379.19, found 402.18 (M + Na)+.
1
44%). H NMR (300 MHz, chloroform-d) δ ppm 1.50 (s, 9 H), 2.14
(dd, J = 13.37, 11.11 Hz, 1 H), 2.73−2.89 (m, 1 H), 2.93−3.09 (m, 1
H), 3.66−3.85 (m, 2 H), 3.91−4.02 (m, 1 H), 4.29 (d, J = 5.65 Hz, 1
H), 4.34−4.48 (m, 2 H), 5.44 (s, 1 H), 7.04 (dd, J = 7.54, 1.13 Hz, 1
H), 7.21−7.42 (m, 10 H), 7.44−7.54 (m, 2 H), 7.70 (d, J = 7.91 Hz, 1
H), 8.15 (s, 1 H). ESI MS m/z: calcd for C31H34N4O5 542.25, found
565.3 (M + 23)+.
N-(2-Aminobenzyl)-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo-
[3,4-a]pyrazine-7(3H)-carboxamide (14). Compound 13 (73 mg,
0.13 mmol) was dissolved in CH2Cl2 (10 mL) at 0 °C. Anhydrous
TFA (5 mL) was then added to the solution and allowed to stir at rt
for 2 h. The resulting solution was basified with sat. aq. NaHCO3 to
pH 11 and extracted with CH2Cl2 (3 × 20 mL). The organic extracts
were combined, dried with MgSO4, and concentrated to provide a
viscous oil. The oil was purified using medium pressure column
chromatography (12 g of SiO2, 100% methylene chloride to 50%
chloroform/methanol/ammonium hydroxide 80/20/1) to provide 14
N-Butyl-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (19). An analogous procedure to that
described for 1 using n-butylisocyanate provided 19 as a viscous oil
(24 mg, 71%). H NMR (300 MHz, chloroform-d) δ ppm 0.87−0.97
1
(m, 3 H), 1.30−1.40 (m, 2 H), 1.41−1.54 (m, 2 H), 2.07−2.19 (m, 1
H), 2.81−2.94 (m, 1 H), 3.07 (td, J = 12.62, 3.39 Hz, 1 H), 3.16−3.27
(m, 2 H), 3.59−3.69 (m, 1 H), 3.83 (dd, J = 13.00, 2.83 Hz, 1 H),
3.95−4.05 (m, 1 H), 4.41 (dd, J = 11.11, 3.58 Hz, 1 H), 4.61 (t, J =
5.27 Hz, 1 H), 7.23−7.44 (m, 8 H), 7.47−7.57 (m, 2 H). ESI HRMS
m/z: calcd for C23H27N3O3 393.21, found 416 0.19 (M + Na)+.
3-Oxo-N-pentyl-1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (20). An analogous procedure to that
described for 1 using n-pentylisocyanate provided 20 as viscous oil (39
1
as a viscous oil (59 mg, 99%). H NMR (300 MHz, chloroform-d) δ
ppm 2.07−2.21 (m, 1 H), 2.79 (td, J = 12.81, 3.77 Hz, 1 H), 2.92−
3.13 (m, 1 H), 3.61−3.81 (m, 3 H), 3.93 (dd, J = 13.19, 3.01 Hz, 3 H),
4.25−4.50 (m, 4 H), 5.06 (br s, 1 H), 6.61−6.72 (m, 2 H), 6.99−7.14
(m, 2 H), 7.23−7.40 (m, 8 H), 7.44−7.52 (m, 2 H). ESI MS m/z:
calcd for C26H26N4O3 442.20, found 443.5 (M + H)+.
1
mg, 64%). H NMR (300 MHz, chloroform-d) δ ppm 0.81−0.98 (m,
3 H), 1.18−1.41 (m, 4 H), 1.49 (quin, J = 7.25 Hz, 2 H), 2.13 (dd, J =
13.19, 11.30 Hz, 1 H), 2.79−2.97 (m, 1 H), 3.07 (td, J = 12.62, 3.77
Hz, 1 H), 3.14−3.31 (m, 2 H), 3.56−3.72 (m, 1 H), 3.83 (dd, J =
13.19, 2.64 Hz, 1 H), 4.01 (ddd, J = 13.37, 3.58, 1.13 Hz, 1 H), 4.41
(dd, J = 11.30, 3.77 Hz, 1 H), 4.57 (t, J = 5.46 Hz, 1 H), 7.16−7.46
(m, 8 H), 7.47−7.61 (m, 2 H). ESI HRMS m/z: calcd for C24H29N3O3
407.22, found 430.21 (M + Na)+.
N-(2-(Methylamino)benzyl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (15). To a solution of 14
(55 mg, 0.124 mmol) in 1,2-dichloroethane (5 mL) was added
formaldehyde (37% solution in water, 37 μL, 0.50 mmol) and sodium
triacetoxyborohydride (105 mg, 0.50 mmol). The reaction mixture was
stirred at room temperature for 1 h and then quenched with NaHCO3
(sat. aq. solution; 10 mL), and the aqueous layer was washed with
methylene chloride (2 × 10 mL). The organic layer was separated,
washed with brine (5 mL), dried (MgSO4), and concentrated under
reduced pressure to provide an oil. The oil was purified by column
chromatography (12 g of SiO2, 100% methylene chloride to 50%
chloroform/methanol/ammonium hydroxide 80/20/1) to provide 15
N-Hexyl-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (21). An analogous procedure to that
described for 1 using n-hexylisocyanate provided 21 as a viscous oil
1
(39 mg, 61%). H NMR (300 MHz, chloroform-d) δ ppm 0.84−0.92
(m, 3 H), 1.25−1.33 (m, 6 H), 1.43−1.53 (m, 2 H), 2.06−2.15 (m, 1
H), 2.89 (dd, J = 13.00, 3.58 Hz, 1 H), 3.05 (dd, J = 12.62, 3.58 Hz, 1
H), 3.16−3.24 (m, 2 H), 3.60−3.69 (m, 1 H), 3.60−3.68 (m, 1 H),
3.83 (dd, J = 13.00, 2.83 Hz, 1 H), 4.01 (ddd, J = 13.38, 3.58, 1.13 Hz,
1 H), 4.41 (dd, J = 11.30, 3.77 Hz, 1 H), 4.59 (s, 1 H), 7.26−7.42 (m,
8 H), 7.47−7.55 (m, 2 H). ESI HRMS m/z: calcd for C25H31N3O3
421.24, found 444.22 (M + Na)+.
1
as a white solid (6 mg, 5%). H NMR (300 MHz, DMSO-d6) δ ppm
2.10−2.25 (m, 1 H), 2.54 (s, 1 H), 2.79 (s, 3 H), 3.19−3.31 (m, 1 H),
3.37−3.47 (m, 1 H), 3.50−3.70 (m, 2 H), 4.32 (d, J = 6.40 Hz, 2 H),
4.43 (s, 2 H), 4.67−4.79 (m, 1 H), 6.66−6.78 (m, 2 H), 6.95−7.14
1
N-(2-(Methylamino)ethyl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (22). Compound 40 (50
mg, 0.14 mmol) was dissolved in THF (15 mL) and methylamine (2
M soln. in THF, 0.70 mL, 1.4 mmol), and NaHCO3 (29 mg, 0.35
mmol), and KI (13 mg, 0.06 mmol) were added. The reaction mixture
was heated to 73 °C for 60 h and concentrated to dryness. The residue
was purified using medium pressure column chromatography (12 g of
SiO2, 100% ethyl acetate to 50% chloroform/methanol/ammonium
(m, 2 H), 7.25−7.48 (m, 8 H), 7.54−7.65 (m, 2 H). H NMR (300
MHz, chloroform-d) δ ppm 2.24 (dd, J = 13.56, 11.30 Hz, 1 H), 2.88
(s, 3 H), 2.91−3.05 (m, 1 H), 3.05−3.23 (m, 1 H), 3.67 (d, J = 12.43
Hz, 2 H), 3.85 (dd, J = 13.00, 2.83 Hz, 1 H), 4.23−4.32 (m, 1 H),
4.38−4.46 (m, 1 H), 4.47−4.61 (m, 3 H), 6.71−6.86 (m, 2 H), 6.96
(d, J = 6.40 Hz, 1 H), 7.11−7.21 (m, 1 H), 7.27−7.42 (m, 8 H), 7.47−
7.56 (m, 2 H). ESI MS m/z: calcd for C27H28N4O3 456.54, found
458.3 (M + H)+.
1
hydroxide 80/20/1) to provide 22 (15 mg, 30%). H NMR (300
N-Ethyl-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]-
pyrazine-7(3H)-carboxamide (16). An analogous procedure to that
described for 1 using ethylisocyanate provided 16 as viscous oil (160
MHz, chloroform-d) δ ppm 1.87 (br s, 1 H), 2.14 (dd, J = 13.19, 11.30
Hz, 1 H), 2.41 (s, 3 H), 2.73 (t, J = 5.65 Hz, 2 H), 2.86 (td, J = 12.81,
739
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
3.77 Hz, 1 H), 3.08 (td, J = 12.72, 3.58 Hz, 1 H), 3.23−3.38 (m, 2 H),
3.67−3.75 (m, 1 H), 3.84 (dd, J = 13.19, 2.64 Hz, 1 H), 4.02 (ddd, J =
13.37, 3.58, 1.13 Hz, 1 H), 4.41 (dd, J = 11.30, 3.77 Hz, 1 H), 5.26−
5.34 (m, 1 H), 7.25−7.42 (m, 8 H), 7.48−7.58 (m, 2 H). ESI HRMS
m/z: calcd for C22H26N4O3 394.20, found 395.21 (M + H)+.
N-(3-(Methylamino)propyl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (23). An analogous
procedure to that described for 22 using 41 as the starting material
provided 23 as a viscous oil (8 mg, 20%). 1H NMR (300 MHz,
chloroform-d) δ ppm 1.62−1.67 (m, 3 H), 2.15 (dd, J = 13.37, 11.11
Hz, 1 H), 2.37 (s, 3 H), 2.70−2.78 (m, 2 H), 2.84 (dd, J = 13.19, 3.39
Hz, 1 H), 3.07 (td, J = 12.62, 3.77 Hz, 1 H), 3.28−3.41 (m, 2 H),
3.67−3.77 (m, 1 H), 3.83 (dd, J = 13.37, 2.83 Hz, 1 H), 3.90−3.98 (m,
1 H), 4.41 (dd, J = 11.30, 3.77 Hz, 1 H), 6.92 (d, J = 4.90 Hz, 1 H),
7.26−7.44 (m, 8 H), 7.48−7.56 (m, 2 H). ESI HRMS m/z: calcd for
C23H28N4O3 408.22, found 409.22 (M + H)+.
N-(3-(Dimethylamino)propyl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (24). An analogous
procedure to that described for 22 using 41 and dimethylamine as
starting materials provided 24 as a viscous oil (19 mg, 37%). 1H NMR
(300 MHz, chloroform-d) δ ppm 1.61−1.68 (m, 3 H), 2.14−2.21 (m,
6 H), 2.37−2.45 (m, 2 H), 2.79−2.87 (m, 1 H), 3.00−3.08 (m, 1 H),
3.28−3.36 (m, 2 H), 3.62−3.73 (m, 1 H), 3.83 (dd, J = 12.81, 2.64 Hz,
1 H), 3.89 (br s, 1 H), 4.40 (dd, J = 11.11, 3.58 Hz, 1 H), 7.16 (br s, 1
H), 7.28−7.44 (m, 8 H), 7.48−7.55 (m, 2 H). ESI HRMS m/z: calcd
for C24H30N4O3 422.23, found 445.22 (M + Na)+.
1,1-Diphenyl-7-(3-(piperidin-1-yl)propanoyl)tetrahydro-1H-
oxazolo[3,4-α]pyrazin-3(5H)-one Trifluoroacetate (25). To a stirred
solution of 3-(piperidin-1-yl)propanoic acid was added a 2-fold excess
of thionyl chloride, and the reaction mixture was stirred at room
temperature for 12 h. The reaction mixture was concentrated to
dryness and redissolved in anhydrous CH2Cl2 (10 mL, 0.04 mM). To
this solution was added 37 (29 mg, 0.1 mmol) and TEA (56 μL, 0.4
mmol). The mixture was allowed to stir at rt for 6 h and was
concentrated to dryness. The residue was purified by column
chromatography (12 g of SiO2, 100% methylene chloride to 50%
chloroform/methanol/ammonium hydroxide 80/20/1) and then
semipreparative HPLC (Synergy Hydro; 15 mL/min 5%−50% B
over 30 min) to afford 25 as viscous oil (20 mg, 37%). 1H NMR (300
MHz, chloroform-d) δ ppm 1.32−1.53 (m, 1 H), 2.04 (t, J = 13.00 Hz,
2 H), 2.48−2.96 (m, 5 H), 2.96−3.19 (m, 3 H), 3.19−3.81 (m, 7 H),
3.92 (d, J = 10.17 Hz, 1 H), 4.37 (t, J = 7.72 Hz, 1 H), 4.48−4.69 (m,
1 H), 7.17−7.70 (m, 10 H), 8.77 (br s, 1 H). ESI MS m/z: calcd for
C26H31N3O3 433.55, found 434 (M + H)+. HPLC (Synergy Hydro, 20
min) tR = 15.52 min (>99%).
N-(1-Methylpiperidin-4-yl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide trifluoroacetate (26).
An analogous procedure to that described for 2 using 1-
methylpiperidin-4-amine provided crude 26, which was purified by
semipreparative HPLC (Synergy Hydro; 15 mL/min 5%−60% B over
30 min) to afford 26 (25 mg, 26%). 1H NMR (300 MHz, chloroform-
d) δ ppm 1.97−2.20 (m, 5 H), 2.75−2.92 (m, 6 H), 3.05 (td, J =
12.62, 3.39 Hz, 1 H), 3.58 (d, J = 12.06 Hz, 2 H), 3.76−4.05 (m, 4 H),
4.38 (dd, J = 11.11, 3.58 Hz, 1 H), 6.52 (br s, 1 H), 7.20−7.44 (m, 8
H), 7.46−7.58 (m, 2 H), 11.56 (br s, 1 H). ESI MS m/z: calcd for
C25H30N4O3 434.54, found 435.2 (M + H)+, 547.7 (M + TFA − H)−.
HPLC (Synergy Hydro, 20 min) tR = 14.47 min (>99%).
solution of 27 (35 mg, 0.7 mmol) in CH2Cl2 (3 mL) was added
trifluoroacetic acid (0.3 mL). The reaction mixture was allowed to stir
at rt for 3 h and was concentrated to dryness. Purification by
semipreparative HPLC (Synergy Hydro; 15 mL/min 5%−60% B over
1
30 min) provided 28 as a white solid (28 mg, 77%). H NMR (300
MHz, DMSO-d6) δ ppm 1.82−1.95 (m, 1 H), 2.06−2.20 (m, 2 H),
2.65−2.78 (m, 1 H), 3.05−3.21 (m, 1 H), 3.27 (dt, J = 11.59, 4.00 Hz,
1 H), 3.33−3.70 (m, 6 H), 3.75 (d, J = 4.52 Hz, 1 H), 4.64 (ddd, J =
10.83, 6.31, 3.58 Hz, 1 H), 7.29−7.48 (m, 8 H), 7.58 (d, J = 7.54 Hz, 2
H), 8.12 (br s, 3 H). ESI MS m/z: calcd for C28H34N4O5 406.49,
found 407.6 (M + H)+. HPLC (Synergy Hydro, 20 min) tR = 13.89
min (>99%).
(3S)-tert-Butyl-3-(3-oxo-1,1-diphenylhexahydro-1H-oxazolo[3,4-
α]pyrazine-7-carboxamido)pyrrolidine-1-carboxylate (29). An anal-
ogous procedure to that described for 2 using (S)-tert-butyl 3-
aminopyrrolidine-1-carboxylate provided 29 as a viscous oil (46 mg,
53%). 1H NMR (300 MHz, chloroform-d) δ ppm 1.39−1.52 (m, 9 H),
2.08−2.23 (m, 2 H), 2.85−3.01 (m, 1 H), 3.02−3.16 (m, 2 H), 3.41
(br s, 2 H), 3.61 (d, J = 5.65 Hz, 2 H), 3.84 (br s, 1 H), 3.94−4.07 (m,
1 H), 4.27−4.38 (m, 1 H), 4.38−4.49 (m, 2 H), 7.27−7.46 (m, 8 H),
7.47−7.57 (m, 2 H). ESI MS m/z: calcd for C28H34N4O5 506.60,
found 407.4 (C28H34N4O5 − Boc + H)+, 507.2 (C28H34N4O5 + H)+.
HPLC (Synergy Hydro, 20 min) tR = 19.48 min (83.4%).
3-Oxo-1,1-diphenyl-N-((S)-pyrrolidin-3-yl)tetrahydro-1H-
oxazolo[3,4-α]pyrazine-7(3H)-carboxamide Trifluoroacetate (30).
An analogous procedure to that described for 28 provided 30 (35
1
mg, 75%). H NMR (300 MHz, chloroform-d) δ ppm 2.05−2.21 (m,
2 H), 2.35 (d, J = 7.16 Hz, 1 H), 2.71−2.92 (m, 1 H), 3.03 (t, J =
12.24 Hz, 1 H), 3.17−3.62 (m, 4 H), 3.84 (t, J = 13.00 Hz, 2 H),
3.95−4.12 (m, 1 H), 4.40 (ddd, J = 11.02, 7.44, 3.77 Hz, 1 H), 4.60 (br
s, 1 H), 7.28−7.43 (m, 8 H), 7.45−7.58 (m, 2 H), 8.78 (br s, 1 H),
9.84 (br s, 1 H). ESI MS m/z: calcd for C28H34N4O5 406.49, found
407.4 (M + H)+, 519.5 (M + TFA − H)−. HPLC (Synergy Hydro, 20
min) tR = 13.97 min (>99%).
(−3aS)-N-(4-Fluorobenzyl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (31). An analogous
procedure to that described for 1 using 38 provided 31 as a colorless
1
semisolid (8.0 mg, 30%). H NMR (300 MHz, chloroform-d) δ ppm
2.17 (dd, J = 13.19, 11.30 Hz, 1 H), 2.83−3.00 (m, 1 H), 3.01−3.16
(m, 1 H), 3.63 (dd, J = 13.00, 1.70 Hz, 1 H), 3.86 (dd, J = 13.00, 2.45
Hz, 1 H), 4.03 (dd, J = 13.19, 2.26 Hz, 1 H), 4.26−4.49 (m, 3 H),
4.69−4.81 (m, 1 H), 6.95−7.09 (m, 1 H), 7.21−7.45 (m, 11 H), 7.47−
7.56 (m, 2 H). tR = 11.47 min, peak 1, Chiralpak-IA 4.6 mm × 250
mm column, in 9:1 hexane/isopropanol at a flow rate of 1 mL/min.
ESI MS m/z: calcd for C26H24FN3O3 445.49, found 446.7 (M + H)+.
20
[α]D = −151 (c = 0.585, CHCl3).
(+3aR)-N-(4-Fluorobenzyl)-3-oxo-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (32). An analogous
procedure to that described for 1 using 39 provided 32 as a colorless
1
semisolid (8 mg, 30%). H NMR (300 MHz, chloroform-d) δ ppm
2.17 (dd, J = 13.19, 11.30 Hz, 1 H), 2.83−3.00 (m, 1 H), 3.01−3.16
(m, 1 H), 3.63 (dd, J = 13.00, 1.70 Hz, 1 H), 3.86 (dd, J = 13.00, 2.45
Hz, 1 H), 4.03 (dd, J = 13.19, 2.26 Hz, 1 H), 4.26−4.49 (m, 3 H),
4.69−4.81 (m, 1 H), 6.95−7.09 (m, 1 H), 7.21−7.45 (m, 11 H), 7.47−
7.56 (m, 2 H); tR = 14.77 min, peak 2, Chiralpak-IA 4.6 mm × 250
mm column, in 9:1 hexane/isopropanol at a flow rate of 1 mL/min.
ESI MS m/z: calcd for C26H24FN3O3 445.49, found 446.7 (M + H)+.
(−3aS)-3-Oxo-1,1-diphenyl-N-(2-(piperidin-1-yl)ethyl)tetrahydro-
1H-oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (33). Compound 33
was isolated from racemic 2 via semipreparative chiral chromatography
on a Chiralpak-IA 20 mm × 250 mm column, utilizing an isocratic
method (95:5:0.1 hexane/isopropanol/diethyl amine) at a flow rate of
12 mL/min (100 mg in 400 μL of ethanol/DMSO), to obtain 7.3 mg
tert-Butyl-1-(3-oxo-1,1-diphenylhexahydro-1H-oxazolo[3,4-α]-
pyrazine-7-carbonyl)pyrrolidin-3-ylcarbamate (27). An analogous
procedure to that described for 2 using tert-butyl pyrrolidin-3-
ylcarbamate provided 27 as a viscous oil (37 mg, 43%). 1H NMR (300
MHz, chloroform-d) δ ppm 1.42−1.48 (m, 9 H), 1.60−1.92 (m, 1 H),
2.02−2.24 (m, 2 H), 2.87 (br s, 1 H), 3.02−3.25 (m, 2 H), 3.34−3.66
(m, 6 H), 3.71−3.90 (m, 2 H), 4.09−4.21 (m, 1 H), 4.55 (ddd, J =
10.83, 6.88, 3.39 Hz, 1 H), 7.28−7.44 (m, 8 H), 7.50−7.57 (m, 2 H).
ESI MS m/z: calcd for C28H34N4O5 506.60, found 507.1 (M + H)+,
505.6 (M − H)−. HPLC (Synergy Hydro, 20 min) tR = 19.45 min
(80%).
20
(tR = 10.45 min, peak 1). [α]D = −142 (c = 0.81, methanol).
(+3aR)-3-Oxo-1,1-diphenyl-N-(2-(piperidin-1-yl)ethyl)tetrahydro-
1H-oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (34). Fractions from
the isolation of compound 33 that were enriched in compound 34
were pooled (49 mg, 95% enantiomeric purity) and rechromato-
graphed utilizing the same column and isocratic method (40 mg in 400
7-(3-Aminopyrrolidine-1-carbonyl)-1,1-diphenyltetrahydro-1H-
oxazolo[3,4-α]pyrazin-3(5H)-one Trifluoroacetate (28). To a
740
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
μL of ethanol/DMSO) to obtain 26.2 mg of pure 34 as a colorless oil
until the solid dissolved and was allowed to stand at room temperature
for 8 h. The resulting crystals were collected and dried under reduced
pressure to afford 38a as colorless needles (0.106 g), mp =211−213
(tR = 12.67 min, peak 2); [α]D = +138 (c = 0.82, methanol).
20
(1,4-Dibenzylpiperazin-2-yl)diphenylmethanol (35). To a solution
of phenyl lithium (74.7 mL, 1.8 M in dibutylether) and THF
(anhydrous, 170 mL) at −78 °C was added dropwise a solution of
ethyl 1,4-dibenzylpiperazine-2-carboxylate (9 g, 26.59 mmol) in THF
(anhydrous, 100 mL). After complete addition, the reaction mixture
was warmed to room temperature over 30 min and then stirred at rt
for 1 h. The reaction mixture was quenched with H2O (200 mL) and
extracted with CH2Cl2 (2 × 250 mL). The organic layer was dried
(MgSO4), concentrated, and purified by medium pressure column
chromatography (80 g of SiO2, 100% CH2Cl2) to provide 35 as a
20
°C; [α]D = −151.43 (c = 0.175, MeOH). Compound 38a was sent
for X-ray analysis.
(+3aR)-1,1-Diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-
one (39). Fractions from the isolation of compound 38 that were
enriched in compound 39 were pooled (126 mg, 93% enantiomeric
purity) and rechromatographed over six separations (16 mg in 100 μL
of ethanol/DMSO per run) utilizing the same column and isocratic
method to obtain 85.7 mg of pure 39 as a colorless oil (tR = 23.4 min,
20
peak 2). [α]D = +252.8 (c = 0.345, MeOH).
1
N-(2-Chloroethyl)-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo[3,4-
a]pyrazine-7(3H)-carboxamide (40). An analogous procedure to that
described for 1 using 2-chloroethyl isocyanate provided 40 as a viscous
yellow solid (11.92 g, quant.). H NMR (300 MHz, chloroform-d) δ
ppm 1.56 (br s, 1 H), 2.32−2.48 (m, 2 H), 2.48−2.61 (m, 2 H), 2.79
(d, J = 11.68 Hz, 1 H), 3.28−3.46 (m, 2 H), 3.53−3.78 (m, 4 H),
6.96−7.35 (m, 16 H), 7.35−7.45 (m, 2 H), 7.45−7.54 (m, 2 H). ESI
MS m/z: calcd for C31H32N2O 448.6, found 449.7 (M + H)+, 448.0
(M − H)−.
1
oil (39 mg, 61%). H NMR (300 MHz, chloroform-d) δ ppm 2.09−
2.26 (m, 1 H), 2.74−2.95 (m, 1 H), 3.08 (td, J = 12.62, 3.77 Hz, 1 H),
3.44−3.68 (m, 4 H), 3.70−3.87 (m, 2 H), 3.98 (dd, J = 13.56, 2.26 Hz,
1 H), 4.42 (dd, J = 11.30, 3.77 Hz, 1 H), 5.33 (t, J = 5.09 Hz, 1 H),
7.18−7.43 (m, 8 H), 7.44−7.60 (m, 2 H).
Diphenyl(piperazin-2-yl)methanol (36). To a solution of 35 (2.54
g, 5.76 mmol) in CH2Cl2 (100 mL) was added 1 N HCl in Et2O (8.5
mL, 8.5 mmol). The reaction mixture was allowed to stir at room
temperature for 1 h and concentrated to dryness, and the residual
white powder was dried under high vacuum for 1 h. The residue was
redissolved in hot ethanol (110 mL) and hydrogenated (46 psi) over
5% Pd/BaSO4 (1 g) and 10% Pd/C (1 g) at rt for 20 h. The reaction
mixture was filtered through Celite, and the filtrate was concentrated
to dryness. The resulting residue was suspended in CH2Cl2 (200 mL)
and washed with aq. NaOH (1 N, 50 mL). The aqueous layer was
extracted with CH2Cl2 (2 × 200 mL); the combined organic layers
were washed with brine (2 × 50 mL), dried (MgSO4), and
concentrated to dryness to provide crude 36 (1.16 g) as a yellow
N-(3-Chloropropyl)-3-oxo-1,1-diphenyltetrahydro-1H-oxazolo-
[3,4-a]pyrazine-7(3H)-carboxamide (41). An analogous procedure to
that described for 1 using 3-chloropropylisocyanate provided 41 as a
1
viscous oil (195 mg, %). H NMR (300 MHz, chloroform-d) δ ppm
1.92−2.02 (m, 2 H), 2.14 (dd, J = 13.56, 11.30 Hz, 1 H), 2.77−2.94
(m, 1 H), 3.06 (td, J = 12.62, 3.77 Hz, 1 H), 3.38 (qd, J = 6.22, 2.83
Hz, 2 H), 3.52−3.64 (m, 2 H), 3.64−3.76 (m, 1 H), 3.83 (dd, J =
13.19, 2.64 Hz, 1 H), 3.97 (ddd, J = 13.37, 3.58, 1.13 Hz, 1 H), 4.41
(dd, J = 11.11, 3.58 Hz, 1 H), 5.00 (t, J = 5.65 Hz, 1 H), 7.24−7.43
(m, 8 H), 7.46−7.55 (m, 2 H).
X-ray Crystallographic Analysis. Single-crystal X-ray diffraction
data on compound 38a was collected using Cu Kα radiation and a
Bruker Platinum-135 CCD area detector. The crystal was prepared for
data collection by coating with high viscosity microscope oil. The oil-
coated crystal was mounted on a micromesh mount (Mitergen, Inc.)
and transferred to the diffractometer, and a data set was collected at
150 K. The 0.205 × 0.010 × 0.005 mm3 crystal was monoclinic in
space group P21, with unit cell dimensions a = 11.5583(6), b =
7.2211(4), c = 16.4478(9) Å and β = 103.333(2)°. Data was 72.7%
complete to 66.79°θ (∼0.83 Å) with an average redundancy of 1.67.
The structure was solved by direct methods and refined by full-matrix
least-squares on F2 values using the programs found in the SHELXTL
suite (Bruker, SHELXTL v6.10, 2000, Bruker AXS Inc., Madison, WI).
Corrections were applied for Lorentz, polarization, and absorption
effects. Parameters refined included atomic coordinates and aniso-
tropic thermal parameters for all non-hydrogen atoms. Hydrogen
atoms on carbons were included using a riding model [coordinate
shifts of C applied to H atoms] with C−H distance set at 0.96 Å. The
final anisotropic full matrix least-squares refinement on F2 with 336
variables and one restraint converged at R1 = 3.85% for the observed
data and wR2 = 9.89% for all data. The absolute configuration was
determined from the X-ray data using the method of Flack.35
Complete information on data collection and refinement is available in
the Supporting Information.
Atomic coordinates for 38a have been deposited with the
Cambridge Crystallographic Data Centre (deposition number
978117). Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK [fax:
+44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk.
In Vitro Functional Assays. Functional determinations, identi-
fication of functional antagonists at the NPSR, utilized RD-HGA16
cells (Molecular Devices), a CHO cell line stably overexpressing the
promiscuous Gq-protein Ga16. Two individual cell lines were created
that stably express each NPSR variant (NPS Ile107 and Asn107). Cells
were loaded with the calcium sensitive dye calcium3 (Molecular
Devices) for 1 h, and compounds were assayed in separate
experiments for intrinsic activity and for the ability to inhibit NPS
activity as measured by calcium mobilization in the FlexStation assay.
All test compounds were evaluated for activity at least three times, and
each experiment was conducted on a different day. Apparent constant
1
solid. H NMR (300 MHz, chloroform-d) δ ppm 1.60 (br s, 2 H),
2.56−2.77 (m, 3 H), 2.83−3.05 (m, 3 H), 3.70 (dd, J = 9.42, 3.39 Hz,
1 H), 4.48 (br s, 1 H), 7.11−7.38 (m, 6 H), 7.49 (dd, J = 8.29, 1.13
Hz, 2 H), 7.57−7.67 (m, 2 H). ESI MS m/z: calcd for C17H20N2O
268.36, found 269.4 (M + H)+, 251.2 (M − HO) +. HPLC tR = 10.57
min (>99%).
1,1-Diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-one
(37). To a solution of 36 (1g, 3.73 mmol) in THF (anhydrous, 100
mL) was added di-t-butyl dicarbonate (2.03 g, 9.32 mmol), NEt3 (1.3
mL, 9.32 mmol), and DMAP (1.13 g, 9.32 mmol). The reaction
mixture was allowed to stir at rt for 2 h, concentrated to dryness, and
redissolved in CH2Cl2 (100 mL). The solution was washed with aq.
sat. NaHCO3 (50 mL) and brine (50 mL), dried (MgSO4), and
concentrated. The resulting semisolid was redissolved in CH2Cl2 (200
mL), and TFA (2.85 mL, 37.3 mmol) was added. The reaction mixture
was stirred at room temperature for 8 h, concentrated to dryness, and
redissolved in CH2Cl2 (100 mL). The solution was washed with aq.
sat. NaHCO3 (2 × 50 mL), dried (MgSO4), and concentrated. The
residue was purified by column chromatography (80 g of SiO2, 100%
methylene chloride to 40% chloroform/methanol/ammonium hydrox-
ide 80/20/1) to provide 37 as a white solid (900 mg, 82% over three
1
steps). H NMR (300 MHz, DMSO-d6) δ ppm 2.38−2.48 (m, 1 H),
2.89−3.09 (m, 1 H), 3.09−3.21 (m, 1 H), 3.21−3.43 (m, 2 H), 3.82
(dd, J = 14.13, 3.96 Hz, 1 H), 4.91 (dd, J = 11.68, 3.39 Hz, 1 H),
7.25−7.61 (m, 10 H), 9.33 (br s, 1 H). ESI MS m/z: calcd for
C18H18N2O2 294.35, found 295.2 (M + H)+. HPLC (Synergy Hydro,
20 min) tR = 13.59 min (95.1%).
(−3aS)-1,1-Diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-
one (38). Compound 38 was isolated from racemic 37 via
semipreparative chiral chromatography on a Chiralpak-IA 20 mm ×
250 mm column, utilizing an isocratic method (95:5:0.1 hexane/
isopropanol/diethyl amine) at a flow rate of 12 mL/min over 13
identical separations (40 mg in 150 μL of ethanol/DMSO per run), to
20
obtain 104.8 mg as a colorless oil (tR = 17.2 min, peak 1); [α]D
−258.5 (c = 0.265, MeOH).
=
(−3aS)-1,1-Diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-
one R-Camphor Sulfonate (38a). Compound 38 (0.088 g, 0.30
mmol) was dissolved in MeOH (15 mL) and (R)-camphor sulfonic
acid (0.069 g, 0.30 mmol) was added. The suspension was warmed
741
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
Ke is determined by running the EC50 curves of the agonist, NPS, with
and without the test compounds and measuring the rightward shift of
the agonist curve. Ke values were calculated using the equation Ke =
[L]/((EC50−/EC50+) − 1) where [L] is the test compound
concentration, EC50 is the EC50 of NPS alone, and EC50 is the
EC50 of NPS in the presence of test compound. Therefore, the Ke
value is independent of the concentration of test compounds. A three-
parameter logistic equation was fit to the concentration response data
with Prism (v5 for Windows, GraphPad Software; San Diego, CA) to
calculate the EC50 and Ke values. NPS EC50 values for all experiments
were in the range of 0.25−1.13 nM at the 107I and 5.1−11.2 nM for
the 107N variant.
Pharmacokinetic analyses were conducted using WinNonlin
(version 5.2; Pharsight Corporation, Mountainview CA). Mean
plasma concentration vs time data for 34 was analyzed using
noncompartmental methods (models 200, WinNonlin) to determine
pharmacokinetic parameter estimates. The following parameters were
estimated: maximum observed plasma concentration and time at
−
+
which the maximum concentration was observed (Cmax and Tmax
,
respectively); terminal elimination rate constant (λz) and terminal half-
life (t1/2); area under the plasma concentration vs time curve (AUC)
to last time point with measurable 34 concentration (AUClast) and
AUC extrapolated to infinity (AUC0‑inf). The concentration−time data
of 34 in the brain were also analyzed similarly. Given the limited
amount of data collected in this study, statistical analyses were not
conducted.
NPS Induced Locomotion. Male C57Bl/6 mice (National Cancer
Institute, Bethesda, MD), age 8−12 weeks, were group housed (four
animals per cage) under controlled conditions (temperature 21
2
°C; relative humidity 50%−60%; 12 h light−dark cycle, lights on 6:00
AM) with free access to food and water. Prior to drug injections, mice
were briefly anesthetized with halothane.
ASSOCIATED CONTENT
* Supporting Information
HPLC analysis and MS of target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
Locomotion of mice was monitored in rectangular plexiglass boxes
(60 cm × 40 cm × 50 cm) and cumulative distance traveled (total
distance in centimeters that the animal traveled during the test) was
measured with an automated activity monitor system (Versamax,
Accuscan, Columbus, OH). Mice were allowed to habituate to the
recording chamber for 60 min. Vehicle or compound 34 (in PBS, 10%
Cremophor EL) were injected ip 10 min before the central
administration of either NPS (1 nmol in 2 μL of PBS, 0.1% BSA)
or vehicle (PBS, 0.1% BSA), as describe in Xu et al.2 Mice were
allowed to recover for 5 min and then placed into the apparatus.
Recording of locomotor activity continued for 90 min.
AUTHOR INFORMATION
Corresponding Author
*Scott P. Runyon. E-mail: srunyon@rti.org. Telephone: 919
316-3842. Fax: 919 541-8868.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Pharmacokinetic Analysis. Male C57Bl/6 mice were purchased
from Charles River Laboratories (Raleigh, NC) at a target weight of
25−30 g corresponding to ages of approximately 8 weeks at receipt.
Mice were dosed at 5 mg/kg with a single dose of 34 as a solution in
2.5% dimethylacetamide, 2.5% Cremophor EL, and 95% phosphate
buffered saline, at a final concentration of 0.5 mg/mL. Blood samples
were collected via cardiac puncture from five mice per time point at
target time points of 15 min, 30 min, 1 h, and 2 h postdose. At
sacrifice, the brain of each animal was removed, weighed, and
immediately flash-frozen using liquid nitrogen within low density
polyethylene vials. Whole brain samples were homogenized using a
Potter−Elvehjem type homogenizer in ethanol/water (50:50, v/v)
solution, which was added in a ratio of 3 mL per gram of brain. A 50
μL aliquot of brain homogenate or plasma was transferred to
microcentrifuge tubes followed by 200 μL of deionized/distilled water
and 1.2 mL of a 10 ng/mL solution of reserpine internal standard in
acetonitrile. The samples were vortexed and centrifuged in a model
5415C microcentrifuge (Eppendorf North America, Westbury NY) at
16000g and transferred to autosampler vials for analysis. Calibration
standards were prepared by combining 40 μL blank plasma or blank
brain homogenate and 10 μL of 34 spiking solution in microcentrifuge
tubes and 1.2 mL internal standard solution in acetonitrile. Sample
analysis was conducted using an Applied Biosystems API 5000 triple
quadrupole mass spectrometer (Foster City Ca) interfaced to Agilent
1100 binary pump and autosampler. A Valco valve and Agilent 1100
isocratic pump were used to divert the first minute of the run to waste
while infusing 50:50 acetonitrile/water makeup solvent to the mass
spectrometer. Chromatography was accomplished using a Phenom-
enex Luna C18 column (50 mm × 2 mm i.d., 5 μm particle size) fitted
with a guard cartridge. Injection volumes were 10 μL. Two mobile
phase solutions were used: solution A was 0.1% formic acid in water,
and solution B was 0.1% formic acid in methanol, and the flow rate
was 0.5 mL/min. Initial chromatographic conditions were 90% A then
decreasing to 5% A over 4 min, then returning to initial conditions for
2 min for column equilibration. Compound 34 was monitored in
positive electrospray mode with an MRM transition of 449.20 →
155.10 with DP = 50, CE = 39, and CXP = 36. Reserpine internals
standard was monitored with a transition of 609.20 → 195.00 with DP
= 231, CE = 41, and CXP = 14. Mass spectrometer parameters were as
follows: CUR = 13, GS1 = 50, GS2 = 50, IS = 4000, TEM = 600 °C,
and CAD = 8.
Funding
This work was supported by a United States National Institute
of Mental Health Research Grant (MH081247-01). The X-ray
crystallographic work was supported by NIDA through
Interagency Agreement No. Y1-DA1101 with the Naval
Research Laboratory (NRL).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We also thank Dr. Stewart Clark, Mr. Keith Warner, and Ms.
Tiffany Langston, for their valuable technical assistance.
■
ABBREVIATIONS
■
ANOVA Analysis of variance; API atmospheric pressure
ionization; cAMP cyclic adenosine monophosphate; CNS
central nervous system; CRF corticotrophin releasing factor;
CSA camphor sulfonic acid; DEA diethylamine; DMSO
dimethyl sulfoxide; GPCR G-protein coupled receptor; HTS
high throughput screen; LC/MS liquid chromatography−mass
spectrometry; LC locus ceruleus; NPS neuropeptide S; NPSR
neuropeptide S receptor; PTSD posttraumatic stress disorder;
TFA trifluoroacetic acid; TLC thin-layer chromatography; VTA
ventral tegmental area
REFERENCES
■
(1) Sato, S. Shintani, Y.; Miyajima, N.; Yoshimura, K., Novel G-
protein coupled receptor protein and DNA thereof. World Patent
Application WO 2002031145 A1, 2002.
(2) Xu, Y. L., Reinscheid, R. K., Huitron-Resendiz, S., Clark, S. D.,
Wang, Z., Lin, S. H., Brucher, F. A., Zeng, J., Ly, N. K., Henriksen, S. J.,
de Lecea, L., Civelli, O., and Neuropeptide, S. (2004) A neuropeptide
promoting arousal and anxiolytic-like effects. Neuron 43 (4), 487−497.
(3) Xu, Y. L., Gall, C. M., Jackson, V. R., Civelli, O., and Reinscheid,
R. K. (2007) Distribution of neuropeptide S receptor mRNA and
742
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
neurochemical characteristics of neuropeptide S-expressing neurons in
the rat brain. J. Comp. Neurol. 500 (1), 84−102.
conditioned reinstatement of cocaine-seeking in the rat. Presented at
the Society for Neuroscience 2007, San Diego, 271.18/Z1.
(19) Badia-Elder, N. E., Henderson, A. N., Bertholomey, M. L.,
Dodge, N. C., and Stewart, R. B. (2008) The effects of neuropeptide S
on ethanol drinking and other related behaviors in alcohol-preferring
and -nonpreferring rats. Alcohol.: Clin. Exp. Res. 32 (8), 1380−1387.
(20) Schmoutz, C. D., Zhang, Y., Runyon, S. P., and Goeders, N. E.
(2012) Antagonism of the neuropeptide S receptor with RTI-118
decreases cocaine self-administration and cocaine-seeking behavior in
rats. Pharmacol., Biochem. Behav. 103 (2), 332−337.
(4) Clark, S. D., Duangdao, D. M., Schulz, S., Zhang, L., Liu, X., Xu,
Y. L., and Reinscheid, R. K. (2011) Anatomical characterization of the
neuropeptide S system in the mouse brain by in situ hybridization and
immunohistochemistry. J. Comp. Neurol. 519 (10), 1867−1893.
(5) Reinscheid, R. K., and Xu, Y. L. (2005) Neuropeptide S and its
receptor: A newly deorphanized G protein-coupled receptor system.
Neuroscientist 11 (6), 532−538.
(6) Domschke, K., Reif, A., Weber, H., Richter, J., Hohoff, C.,
Ohrmann, P., Pedersen, A., Bauer, J., Suslow, T., Kugel, H., Heindel,
W., Baumann, C., Klauke, B., Jacob, C., Maier, W., Fritze, J., Bandelow,
B., Krakowitzky, P., Rothermundt, M., Erhardt, A., Binder, E. B.,
Holsboer, F., Gerlach, A. L., Kircher, T., Lang, T., Alpers, G. W.,
Strohle, A., Fehm, L., Gloster, A. T., Wittchen, H. U., Arolt, V., Pauli,
P., Hamm, A., and Deckert, J. (2010) Neuropeptide S receptor gene
 converging evidence for a role in panic disorder. Mol. Psychiatry 16,
938−948.
(7) Okamura, N., Hashimoto, K., Iyo, M., Shimizu, E., Dempfle, A.,
Friedel, S., and Reinscheid, R. K. (2007) Gender-specific association of
a functional coding polymorphism in the Neuropeptide S receptor
gene with panic disorder but not with schizophrenia or attention-
deficit/hyperactivity disorder. Prog. Neuropsychopharmacol. Biol.
Psychiatry 31 (7), 1444−1448.
(8) Raczka, K. A., Gartmann, N., Mechias, M. L., Reif, A., Buchel, C.,
Deckert, J., and Kalisch, R. (2010) A neuropeptide S receptor variant
associated with overinterpretation of fear reactions: a potential
neurogenetic basis for catastrophizing. Mol. Psychiatry 15, 1067−1074.
(9) Leonard, S. K., Dwyer, J. M., Sukoff Rizzo, S. J., Platt, B., Logue,
S. F., Neal, S. J., Malberg, J. E., Beyer, C. E., Schechter, L. E.,
Rosenzweig-Lipson, S., and Ring, R. H. (2008) Pharmacology of
neuropeptide S in mice: therapeutic relevance to anxiety disorders.
Psychopharmacology (Berlin) 197 (4), 601−611.
(10) Rizzi, A., Vergura, R., Marzola, G., Ruzza, C., Guerrini, R.,
Salvadori, S., Regoli, D., and Calo, G. (2008) Neuropeptide S is a
stimulatory anxiolytic agent: a behavioural study in mice. Br. J.
Pharmacol. 154, 471−479.
(11) Vitale, G., Filaferro, M., Ruggieri, V., Pennella, S., Frigeri, C.,
Rizzi, A., Guerrini, R., and Calo, G. (2008) Anxiolytic-like effect of
neuropeptide S in the rat defensive burying. Peptides 29 (12), 2286−
2291.
(12) Gottlieb, D. J., O’Connor, G. T., and Wilk, J. B. (2007)
Genome-wide association of sleep and circadian phenotypes. BMC
Med. Genet. 8 (Suppl 1), No. S9.
(13) Laitinen, T., Polvi, A., Rydman, P., Vendelin, J., Pulkkinen, V.,
Salmikangas, P., Makela, S., Rehn, M., Pirskanen, A., Rautanen, A.,
Zucchelli, M., Gullsten, H., Leino, M., Alenius, H., Petays, T.,
Haahtela, T., Laitinen, A., Laprise, C., Hudson, T. J., Laitinen, L. A.,
and Kere, J. (2004) Characterization of a common susceptibility locus
for asthma-related traits. Science 304 (5668), 300−304.
(14) Cline, M. A., Godlove, D. C., Nandar, W., Bowden, C. N., and
Prall, B. C. (2007) Anorexigenic effects of central neuropeptide S
involve the hypothalamus in chicks (Gallus gallus). Comp. Biochem.
Physiol., Part A: Mol. Integr. Physiol. 148 (3), 657−663.
(21) Cardenas, A., Gines, P., and Runyon, B. A. (2009) Is albumin
infusion necessary after large volume paracentesis? Liver Int. 29 (5),
636−640, () 640−641 discussion.
(22) Si, W., Aluisio, L., Okamura, N., Clark, S. D., Fraser, I., Sutton, S.
W., Bonaventure, P., and Reinscheid, R. K. (2010) Neuropeptide S
stimulates dopaminergic neurotransmission in the medial prefrontal
cortex. J. Neurochem 115, 475−482.
(23) Mochizuki, T., Kim, J., and Sasaki, K. (2010) Microinjection of
neuropeptide S into the rat ventral tegmental area induces hyper-
activity and increases extracellular levels of dopamine metabolites in
the nucleus accumbens shell. Peptides 31 (5), 926−931.
(24) Paneda, C., Huitron-Resendiz, S., Frago, L. M., Chowen, J. A.,
Picetti, R., de Lecea, L., and Roberts, A. J. (2009) Neuropeptide S
reinstates cocaine-seeking behavior and increases locomotor activity
through corticotropin-releasing factor receptor 1 in mice. J. Neurosci.
29 (13), 4155−4161.
(25) Kallupi, M., Cannella, N., Economidou, D., Ubaldi, M., Ruggeri,
B., Weiss, F., Massi, M., Marugan, J., Heilig, M., Bonnavion, P., de
Lecea, L., and Ciccocioppo, R. (2010) Neuropeptide S facilitates cue-
induced relapse to cocaine seeking through activation of the
hypothalamic hypocretin system. Proc. Natl. Acad. Sci. U. S. A. 107
(45), 19567−19572.
(26) Okamura, N., Habay, S. A., Zeng, J., Chamberlin, A. R., and
Reinscheid, R. K. (2008) Synthesis and pharmacological in vitro and in
vivo profile of 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-
7-carboxylic acid 4-fluoro-benzylamide (SHA 68), a selective
antagonist of the neuropeptide S receptor. J. Pharmacol. Exp. Ther.
325 (3), 893−901.
(27) Zhang, Y., Gilmour, B. P., Navarro, H. A., and Runyon, S. P.
(2008) Identifying structural features on 1,1-diphenyl-hexahydro-
oxazolo[3,4-a]pyrazin-3-ones critical for Neuropeptide S antagonist
activity. Bioorg. Med. Chem. Lett. 18 (14), 4064−4067.
(28) Melamed, J. Y., Zartman, A. E., Kett, N. R., Gotter, A. L., Uebele,
V. N., Reiss, D. R., Condra, C. L., Fandozzi, C., Lubbers, L. S., Rowe,
B. A., McGaughey, G. B., Henault, M., Stocco, R., Renger, J. J.,
Hartman, G. D., Bilodeau, M. T., and Trotter, B. W. (2010) Synthesis
and evaluation of a new series of Neuropeptide S receptor antagonists.
Bioorg. Med. Chem. Lett. 20 (15), 4700−4703.
(29) Trotter, B. W., Nanda, K. K., Manley, P. J., Uebele, V. N.,
Condra, C. L., Gotter, A. L., Menzel, K., Henault, M., Stocco, R.,
Renger, J. J., Hartman, G. D., and Bilodeau, M. T. (2010) Tricyclic
imidazole antagonists of the Neuropeptide S Receptor. Bioorg. Med.
Chem. Lett. 20 (15), 4704−4708.
(30) Micheli, F., Di Fabio, R., Giacometti, A., Roth, A., Moro, E.,
Merlo, G., Paio, A., Merlo-Pich, E., Tomelleri, S., Tonelli, F.,
Zarantonello, P., Zonzini, L., and Capelli, A. M. (2010) Synthesis
and pharmacological characterization of 5-phenyl-2-[2-(1-
piperidinylcarbonyl)phenyl]-2,3-dihydro-1H-pyrrolo[1,2-c]imidazo l-
1-ones: a new class of Neuropeptide S antagonists. Bioorg. Med.
Chem. Lett. 20 (24), 7308−7311.
(15) Cline, M. A., Prall, B. C., Smith, M. L., Calchary, W. A., and
Siegel, P. B. (2008) Differential appetite-related responses to central
neuropeptide S in lines of chickens divergently selected for low or high
body weight. J. Neuroendocrinol 20 (7), 904−908.
(16) Meis, S., Bergado-Acosta, J. R., Yanagawa, Y., Obata, K., Stork,
O., and Munsch, T. (2008) Identification of a neuropeptide S
responsive circuitry shaping amygdala activity via the endopiriform
nucleus. PLoS One 3 (7), No. e2695.
(31) Thorsell, A., Tapocik, J. D., Liu, K., Zook, M., Bell, L., Flanigan,
M., Patnaik, S., Marugan, J., Damadzic, R., Dehdashti, S. J., Schwandt,
M. L., Southall, N., Austin, C. P., Eskay, R., Ciccocioppo, R., Zheng,
W., and Heilig, M. (2013) A novel brain penetrant NPS receptor
antagonist, NCGC00185684, blocks alcohol-induced ERK-phosphor-
ylation in the central amygdala and decreases operant alcohol self-
administration in rats. J. Neurosci. 33 (24), 10132−10142.
(17) Jungling, K., Seidenbecher, T., Sosulina, L., Lesting, J., Sangha,
S., Clark, S. D., Okamura, N., Duangdao, D. M., Xu, Y. L., Reinscheid,
R. K., and Pape, H. C. (2008) Neuropeptide S-mediated control of fear
expression and extinction: Role of intercalated GABAergic neurons in
the amygdala. Neuron 59 (2), 298−310.
(18) Cioccioppo, R., Economidou, D., Cannella, N., Braconi, S.,
Stopponi, S. (2007) Neuropeptide s system activation facilitates
(32) Knolker, H. J., and Braxmeier, T. (1998) Isocyanates. Part 5.
̈
Synthesis of chiral oxazolidin-2-ones and imidazolidin-2-ones via
743
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744

ACS Chemical Neuroscience
Research Article
DMAP-catalyzed isocyanation of amines with di-tert-butyl dicarbonate.
Tetrahedron Lett. 39 (51), 9407−9410.
(33) Roy, D., Ducher, F., Laumain, A., and Legendre, J. Y. (2001)
Determination of the aqueous solubility of drugs using a convenient
96-well plate-based assay. Drug Dev. Ind. Pharm. 27 (1), 107−109.
(34) Trapella, C., Pela, M., Del Zoppo, L., Calo, G., Camarda, V.,
Ruzza, C., Cavazzini, A., Costa, V., Bertolasi, V., Reinscheid, R. K.,
Salvadori, S., and Guerrini, R. (2011) Synthesis and separation of the
enantiomers of the neuropeptide S receptor antagonist (9R/S)-3-oxo-
1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-
fluoro-benzylamide (SHA 68). J. Med. Chem. 54 (8), 2738−2744.
(35) Flack, H. D. (1983) On Enantiomorph-Polarity Estimation. Acta
Crystallogr. A39, 876−881.
744
dx.doi.org/10.1021/cn500113c | ACS Chem. Neurosci. 2014, 5, 731−744